(1) Field of the Invention
The present invention relates to a procedure for solid phase peptide synthesis (SPPS), following a linear or convergent strategy, wherein the peptides contain the sequence of general formula (I):Y—A1—A2—A3— . . . A(n-1)—An—X  (I) as well as all pharmaceutically acceptable salts for the peptide formed by the addition of acids or complexes thereof, wherein:                30>n>1        Y=H—, (C1-C20) alkyl or (C1-C20) acyl;        X=—OH, —NHCH3, —NHCH2CH3, —NH—CaHb (10>a>2 and 18>b>5), —NHCF3, —NHCH2CF3, —NH—CaHbFf (10>a>2, 18>b>5, 9>f>0), —NH—CcHdOe (c, d and e>1) or —NH—NR—CO—NH2 (where R represents the side chains of any amino acid);        An (30>n>1) can be any natural of synthetic amino acid, including pyroglutamic acid, provided the sequence contains a minimum of one amino acid with the side chain acylated or alkylated, with a tert-butyl or tert-butoxycarbonyl group for example;using, should it be necessary, temporary protector groups labile to treatments with basic solutions for the Nα groups of the amino acids during synthesis.        
(2) Description of Related Art
The procedure of the invention, the result of a combination of protocols, is characterised by the type of polymeric supports used which are insoluble in organic solvents and which provide a peptide resin bond which is labile in acid solutions of type 1 (see table 1), such as resins of the chlorotrityl, trityl, methyltrityl or methoxytrityl type for example. Said procedure is also characterised by the type of side chain protectors, labile or hyperlabile to treatment with acid, used for the different tri-functional amino acids that the sequence contains. The different degrees of lability of said protectors of the side group with respect to type 1 and/or type 2 acidolytic treatments (see table 1) allow said protectors to be eliminated or maintained in selective fashion.
TABLE 1TypeAcid Solutions1Acetic acid in proportions ranging from 1 to 30% in varioussolvents, or hexafluoroisopropanol in variable concentrations indifferent solvents or equivalent acid solutions.2Acetic acid in proportions greater than 30% in different solvents, ortrifluoroacetic acid in proportions lower than 5% in differentsolvents or equivalent acid solution
In accordance with the invention, the peptides of general formula (I) are synthesised using SPPS by a procedure characterised by the use of a polymeric support which allows the peptide to be cleaved from the peptidyl-resin using type 1 acid solutions (see table 1). Said procedure is also characterised by the use of labile protectors and hyperlabile protectors which can be selectively removed by treatments of with acids of type 1 or 2 (see table 1).
Thus, for the side chain of those tri-functional amino acids in which said chain will be alkylated or acylated in the final product, protectors stable to treatment with acids of type 1 or 2 are used. On the other hand, for the side chain of at least one of the tri-functional amino acids, which has the chain free in the final product, hyperlabile groups are used (labile to treatments with acids of type 2, but stable to treatment with acids of type 1, see table 1) as those used for breaking the peptide-resin bond. This characteristic of the aforementioned protectors allows peptides of general formula I (X≠OH) to then be obtained by derivatisation at the C-terminus, with very good yields, of synthetic intermediates that may have all or some of the side chains of the tri-functional residues protected.
The abbreviations used in the present description have the following meanings:    AcOH: acetic acid    Arg: L-arginine    AzaGly: Azaglycine    Boc: tert-butoxycarbonyl    Bzl: benzyl    ClTrt: Chloro-trityl    DCM: dichloromethane    DIEA: N,N′-diisopropylethylamine    DIPCDI: diisopropylcarbodyimide    DMF: N,N-dimethylformamide    D-Ser: D-serine    Fmoc-aa: Fmoc-amino acid    Fmoc: 9-fluorenylmethoxycarbonyl    His: L-histidine    HOBT: 1-hydroxybenzotriazol    HPLC: high performance liquid chromatography    Ile: L-isoleucine    Leu: L-leucine    LH-RH: Luteinising hormone release hormone    Mmt: Methoxytrityl    NO2: nitro    Pip: piperidine    Pro: L-proline    PyBOP: Benzotriazol-1-yl-oxy hexaflourophosphate    Pyr: pyroglutamic acid    Ser: L-serine    SPPS: Solid phase peptide synthesis    t-Bu: Tert-butyl    TFA: trifluoroacetic acid    Trp: L-tryptophane    Trt: Trityl    Try: L-tyrosine    Z: benzyloxycarbonylThe synthesis methods, described in the state of the art, for the peptides that retain the side chain of at least a tri-functional residue acylated or akylated in the final product, can be divided into two strategies. The first of these strategies condenses free peptide fragments or protected fragments in solution, using classical peptide synthesis schemes well known in the art as well as temporary groups labile to hydrogenolysis and protectors of side chains also labile to hydrogenolysis. The second of the strategies is based on the use of SPPS procedures, employing different polymeric supports and protector groups. One of these strategies is characterised because the synthesis is carried out without protection for the side chains of the tri-functional amino acids that are free of derivatisation in the final products.
The main difficulty involved in the synthesis of compounds of formula (I) lies in the simultaneous presence of residues that should retain their protectors and other residues whose chains should be free in the final product. The most conflictive case is when the peptide contains two or more residues repeated, some of which contain the alkylated or acylated side chain in the final product while others should remain free of alkylation or acylation in the final product.
We shall now review the methods described in the state of the art for the synthesis of products of general formula (1), in particular, gosereline and busereline.
1. Synthesis in Solution, Condensation of Fragments
The patents U.S. Pat. No. 4,100,274 and U.S. Pat. No. 4,024,248 describe methods for synthesis of gosereline and busereline, respectively, by means of condensation of fragments in solution. Both methods are based on the coupling of previously synthesised peptide fragments, mostly with unprotected side chains (see FIG. 1 and FIG. 2), to arrive at the final product with very low yields (lower than 5% with respect to the starting materials) according to that deduced from the examples presented in the specification of said patents.
The method described in U.S. Pat. No. 4,100,274 obtains gosereline by means of the condensation of three pre-formed fragments which contain —NO2 as the protecting group for arginine and -Bzl as the protecting group for tyrosine, both of which are labile to hydrogenolysis. In this method the azaglycine residue is introduced into the C-terminus tripeptide (H-Arg(NO2)Pro-AzGly is synthesised) which is then coupled to Z-Tyr(Bzl)-D-Ser(tBu)-Leu-N3, to give a fragment which, once the Z group is removed, couples to Pyr-His-Trp-Ser-N3 to give gosereline. This last reaction is carried out with all the side chains unprotected with the exception of that belonging to D-Ser(tBu) which possibly leads to the appearance of numerous products with acylation of side chains which must then be hydrolysed with the corresponding reduction in yield.
The literature [Frederich A., Jager G., Radschmit K., Ullman R., Peptides 1992 (Schneider C. H. and Eberle A. E. Eds) 1993 Escom Science Publishers (The Netherlands)] as well as the patent U.S. Pat. No. 4,024,248 describe a method for obtaining busereline (see FIG. 2) analogous to that described for gosereline in U.S. Pat. No. 4,100,274. The only difference lies in the fact that in this case the first fragment to be synthesised has an ethylamide terminus instead of the azaGly residue [H-Arg(NO2)-Pro-NH—CH2CH3].
The patent ES458691, belonging to the same family as the patents U.S. Pat. No. 4,100,274 and U.S. Pat. No. 4,024,248 discloses a method for synthesis of gosereline and busereline in solution by condensation of fragments analogous to those disclosed in the aforementioned American patents. Said patent uses a scheme of side-chain protectors for the tri-functional amino acids which is based on the different lability thereof with respect to hydrogenolysis.
2. Solid-Phase Synthesis
There are three patents that disclose solid phase peptide synthesis (SPPS) methods.
The patent EP 0518656A2 carries out the synthesis of Gosereline on a OBzl-polystyrene resin which is labile to hydrazine, obtaining a derivatization of the hydrazide type during the breakage of the peptide-resin bond at the C-terminus which can later be transformed into the aza-glycine terminus residue. Said procedure uses the tert-butoxycarbonyl and fluorenylmethoxycarbonyl groups as temporary protectors of the Nα group of the amino acids, while for the side chains of the amino acids the following protector groups are used: BrZ for Tyr, Fmoc for His and tBu for D-Ser at the 6 position avoiding the protection of the Ser at position 4 to then convert the hydrazide end into AzaGly-NH2 by reaction KCN after breakage of the peptide-resin bond and deprotection of the side chains. The examples described in the specification do not permit an objective evaluation of the yield that is obtained from the process.
On the other hand, the patent EP 0518655A2 carries out a solid-phase synthesis using a linear strategy starting with the coupling on a peptidyl-resin derivatised with the bi-functional spacer (Rink amide) of AzaGly with no protection for the Tyr and Ser side chains at the 4 position. This process requires that the final peptide be treated with hydrazine to hydrolyse possible side products with acylated amino-acid side chains which are incorporated in free form. This procedure provides an overall yield after purification of 30% according to that deduced from the examples described in the specification of said patent.
The European patent EP 0475184A1 discloses a method for the synthesis of different peptides, among which gosereline can be found, based on the synthesis in solution of Boc-Pro-AzaGly-Bifunctional spacer which is coupled to a polymeric support. With the peptidyl-resin obtained the synthesis continues following a linear strategy with Fmoc-t-Bu strategy. The final step consists of deprotecting the side chains with TFA:ethandithiol (90:10, v/v).
The procedure object of the present invention allows, among other things, the peptides disclosed in the aforementioned patents to be produced with better yields and provides the following synthesis methods that can easily be scaled up, from a common precursor that can be synthesised in bulk.
The appearance during the 90s of hyperlabile protectors allows the synthesis of said derivatives according to an innovative method which is advantageous in that:                The growth of the peptide chain is carried out in solid phase following a completely linear strategy, with better yields than those described up until present and which allows totally or partially protected (X=OH) peptides to be obtained which may be derivatised at the C-terminus with very good yields and using not very aggressive methods common in the art and which do not lead to the formation of large quantities of epimerisation side products.        Elimination of the protectors from the side chain of the tri-functional amino acids is carried out using methods, orthogonal to those used for breaking the peptide-resin bond, with excellent yields and which are less aggressive that those described up until present (treatments with type-2 acid solutions compared to hydrogenolysis or treatments with hydrazine described previously). This fact helps in the industrial scale-up of the process.        